I. Field
The present invention provides methods for measuring the inherent stability of intrachain disulphide-containing domains (e.g., antibody variable domains) and for optimizing the positioning of intrachain disulphide-containing domains within a protein (e.g., a multispecific binding protein, e.g., a DVD-Ig). Also provided are methods of making multispecific binding proteins (e.g., DVD-Ig molecules) comprising two or more antibody variable domains in which the antibody variable domains are optimally positioned within the multispecific binding proteins to enhance stability of the multispecific binding protein. Multispecific binding proteins optimized using the methods disclosed herein are also provided.
II. Description of Related Art
A wide variety of multispecific binding protein formats have been developed (see Kriangkum, J., et al., Biomol Eng, 2001. 18(2): p. 31-40). Amongst them, tandem single-chain Fv molecules and diabodies, and various derivatives thereof, are the most widely used formats for the construction of recombinant bispecific antibodies. More recently diabodies have been fused to Fc to generate more Ig-like molecules, named di-diabodies (see Lu, D., et al., J Biol Chem, 2004. 279(4): p. 2856-65). In addition, multispecific antibody construct comprising two Fab repeats in the heavy chain of an IgG and capable of binding four antigen molecules has been described (see WO 0177342A1, and Miller, K., et al., J Immunol, 2003. 170(9): p. 4854-61).
Despite the many multispecific binding proteins formats available to the skilled artisan, the inherent stability of these molecules is unpredictable. This unpredictability makes the task of generating therapeutically useful multispecific binding proteins onerous and time consuming. Accordingly, there is a need in the art for improved methods of designing, analyzing, optimizing and producing multispecific binding proteins.